Objectives of this project are to (1) use molecular and classical approaches to investigate pathogen-arthropod interactions of vector-borne diseases of human importance in the United States, (2) examine basic biological questions concerning the behavior of ticks and fleas that influence their ability to transmit infectious agents, and (3) to develop rapid and definitive diagnostic assays to detect and identify bacterial pathogens in their arthropod vector. We applied the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery et al. to rapidly identify (within 24 h) rickettsial serotypes in naturally infected ticks. Unlike previously described methods, our PCR assays identify serotypes directly from tick tissues without first isolating the organisms. Thirteen of 226 (5.8%) adult ticks (Dermacentor andersoni) collected in the Bitter Root Mountains of western Montana were positive by hemolymph tests using Gimenez stain. These 13 ticks were analyzed by the above PCR/RFLP assay using two different primer sets and restriction enzyme digests. The first primer set (RpCS) reportedly amplifies DNA from all Rickettsia serotypes tested to date except R. tsutsugamushi serotypes. A second primer set (Rr190) amplified target sequences from a subset of spotted fever group rickettsiae that includes all SFG serotypes investigated to date, except R. akari and R. australis. PCR assays performed using the RpCS primer set resulted in amplification of fragments of the predicted size from 9 of the 13 hemolymph test-positive tick samples. Only 4 of the same 13 tick samples were positive in similar PCR assays performed with the spotted fever group-specific (Rr190) primer set. These same 4 tick samples also gave positive results for PCR assays using the RpCS primer set and RFLP analyses indicated these ticks were infected with R. rickettsii (1 sample) and R. rhipicephali (3 samples). Five of the 9 PCR-positive tick samples were positive only in PCR assays performed with the RpCS primer set. RFLP analyses of PCR fragments amplified from tissues of these 5 ticks indicated they all were infected with R. bellii. We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results. We tested multiple isolates of R. montana (9 isolates), R. bellii (5 isolates), R. rickettsii (Hlp-like) (4 isolates), and R. canada (2 isolates) and found no variation in PCR/RFLP patterns between members of the same serotypes. However, among the 5 isolates of R. rhipicephali tested, two distinctive RFLP patterns were noted for RsaI digests of PCR fragments amplified using primers for the 190 kilodalton antigen gene of R. rickettsii. Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsial serotypes directly in ticks.